My research group has worked for over two decades in the laboratory and clinic to develop safe and effective gene therapies directed at hematopoietic stem and progenitor cells (HSPC). In the rhesus model, shown to be the only predictive assay for human clinical results, we have focused on optimizing gene transfer to primitive stem and progenitor cells, and on understanding and enhancing safety of established and new vector systems. We retrieve and analyze clonal contributions to peripheral blood populations following transplantation of CD34+ transduced progenitor cells. Given the occurence of leukemia in patients receiving gene therapy for severe immunodeficiencies with retrovirally-transduced hematopoietic stem cells, we have performed large scale sequencing of retroviral insertion sites in rhesus macaques transplanted with cells transduced either with MLV, HIV or SIV vectors, and we continue to follow animals transplanted up to 18 years ago with transduced CD34+ cells, a unique resource for predicting the long-term safety and utility of retroviral gene transfer. We have applied our genetic barcoding technology to map contributions of thousands of individual hematopoietic stem and progenitor cell clones, and investigated whether clonal expansion as an early measure of genotoxicity can be assessed in a high throughput manner using this approach. Relevant preclinical model for assessing genotoxicity prior to clinical trials are an unmet need, since in vitro assays and murine models have not been predictive. The quantitative assessment of oligoclonality in vivo, via our highly sensitive and quantitative barcoding approach, allows relevant comparisons between vectors. In animals followed for 1-5.5 years, we have now clonally tracked the behavior of almost 100,000 lentivirally-transduced HSPC over time, and whether the vector contains strong (SFFV), medium (MSCV) or weak promoter/enhancers, with a single exception we have not seen clonal expansion or other evidence of genotoxicity. However recently we have encountered the first clear evidence of genotoxicity utilizing a lentiviral vector to transduce HSPC in a human or primate. A rhesus macaque receiving lentivirally-barcoded cells, with a vector containing a moderately strong enhancer, developed markedly abnormal hematopoiesis, with profound thrombocytopenia, eosinophilia, and most strikingly an erythroid expansion with very high levels of circulating nucleated red blood cells. These abnormal cell populations were shown to be clonal by barcode retrieval, and the clone contains 9 independent barcoded insertions. We have retrieved the insertion sites, and analysis of which site or sites is most likely responsible for the syndrome is ongoing. This important information is being presented at an international meeting soon and will also be transmitted to the FDA. Given the potential for genotoxicity with random integration of lentiviral vectors, and other drawbacks of gene addition as compared to targeted gene correction approaches, we have utilized the rhesus macaque to explore CRISPR/Cas9 genome editing to create disease models and to develop gene editing therapies targeting HSPC. We have optimized CRISPR/Cas9 gene editing of rhesus CD34+ HSPC, initially knocking out loci and creating indels via non-homologous end joining repair. We have successfully engrafted 6 animals with gene-edited cells, with long-term engraftment at levels of up to 10% or more for cells that have targeted indels. We have begun to create a macaque model of paroxysmal nocturnal hemoglobinuria by targeting the PIG-A locus, we have knocked out CD33 in neutrophils produced from edited HSPC as an approach to make marrow resistant to CAR-T cells targeting CD33 in acute myeloid leukemia, and we have created a robust macaque model of CHIP by targeting DNMT3, TET2 and ASXL1 with CRISPR/Cas9 mediated editing to create loss of function mutations. We have shown marked clonal expansion of TET2 mutated clones but not DNMT2 or ASXL1 to date. We have multiple ongoing studies to investigate the biology of clonal expansion in these animals, and to utilize our CD33 KO animals for CAR-T cell therapy development. We are also collaborating with Shengdar Tsai and Keith Joung to validate various approaches to identifying and detecting off-target effects of CRISPR/Cas9 in HSPC and their progeny.